Bioactivity, pharmacokinetics, and immunogenicity assays in preclinical and clinical trials for recombinant human endostatin.
OBJECTIVE
To determine the in vitro and in vivo bioactivity of recombinant human endostatin (rhEndostatin) and to analyze its pharmacokinetics and immunogenicity in rhesus monkeys and patients.


METHODS
The physical chemical characteristics of rhEndostatin were detected according to Pharmacopoeia of the People's Republic of China (2005 edition, part III). Its in vitro and in vivo bioactivities were assayed via proliferation-inhibition on human umbilical vein endothelial cells and their inhibitory effect on tumor-bearing mice models. Serum concentrations of rhEndostatin in monkeys and patients were determined by an enzyme immunoassay method.


RESULTS
The corresponding specific in vitro activities of rhEndostatin obtained from the cell counting method, 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and lactate dehydrogenase assay, respectively, were 6.4 x 10(7), 6.7 x 10(7), and 3.8 x 10(8) U/mg, and the in vivo antitumoral potency was 4.04 x 10(7) U/mg. In rhesus monkeys, there were no gender differences in all pharmacokinetic parameters. Serum anti-rhEndostatin immunoglobulin (Ig)G antibodies were generated quickly after intravenous (iv) administration and decreased rapidly when therapy was stopped. In phase I clinical trials, linearity in the pharmacokinetics of rhEndostatin was indicated by dose-proportionate increases in the area under the curve and the maximum serum concentration. Serum rhEndostatin reached a steady-state level after 7 d of successive administration with the average concentration at a steady state of 272.44+/-91.98 ng/mL. Neither IgG nor IgM antibodies against rhEndostatin were observed in patients.


CONCLUSIONS
RhEndostatin exhibited a definite proliferation- inhibition effect on HUVEC, and significant antitumoral activity in mice. The immunoreactivity of rhesus monkeys to rhEndostatin is common, and rhEndostatin showed no immunogenicity in patients in this trial. The results provide a basis for further clinical trials.
Hu
Bi
B
Department of Biological Science and Technology and State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, China.
Zhu
Hao-wen
HW
Zhu
Li-ping
LP
Li
Chen
C
Rong
Zhi-gang
ZG
Xu
Jia-ming
JM
Wu
Zhi-wei
ZW
Wang
Jian-jun
JJ
Xu
Gen-xing
GX
D020533:Angiogenesis Inhibitors / Q000276:immunology / Q000493:pharmacokinetics / Q000627:therapeutic use*; D000818:Animals; D000970:Antineoplastic Agents / Q000276:immunology / Q000493:pharmacokinetics / Q000627:therapeutic use*; D043169:Endostatins / Q000276:immunology / Q000493:pharmacokinetics / Q000627:therapeutic use*; D042783:Endothelial Cells / Q000187:drug effects; D005260:Female; D006801:Humans; D008253:Macaca mulatta; D008297:Male; D051379:Mice; D011994:Recombinant Proteins / Q000276:immunology / Q000493:pharmacokinetics / Q000627:therapeutic use; D012727:Sex Characteristics; D013778:Tetrazolium Salts; D013844:Thiazoles
D020533:Angiogenesis Inhibitors; D000970:Antineoplastic Agents; D043169:Endostatins; D011994:Recombinant Proteins; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue